PROJECT SUMMARY Long QT Syndrome (LQTS) is an acquired or inherited disorder, characterized by prolonged QT interval, exertion-triggered arrhythmias, and sudden cardiac death. The most prevalent hereditary LQTS subtype, LQT1, results from loss-of-function mutations in the KCNQ1, a voltage-gated potassium channel that, in combination with the auxiliary subunit KCNE1, is critical for cardiac repolarization. There is growing consensus in LQT1, as with many ion channelopathies, that a majority of mutations result from reduced expression of channel subunits at the surface membrane. In contrast, the tools available to probe trafficking deficiencies and elucidate mechanistic insight remain limited and underdeveloped, hindering the progress of new potential therapeutic strategies. Ubiquitin has emerged as an important post-translational signal with diverse roles in sorting, signaling, and degradation. Previous over-expression studies have highlighted the role of the E3 ubiquitin ligase NEDD4L in the regulation of Q1 surface trafficking and stability. Nevertheless, the inability to selectively investigate these processes in the native cellular environment have left behind many fundamental unknowns relating to ubiquitin regulation of Q1 and its contributions to LQT1. In this proposal, I look to address this critical gap in two parallel, complementary aims. First, I will develop engineered E3 ubiquitin ligases and deubiquitinases (DUBs) to precisely and specifically manipulate the ubiquitination pattern of KCNQ1 subunits in the cellular context. Utilizing mass spectrometric approaches, I will further interrogate the molecular code responsible for divergent cell biological process of Q1 trafficking and stability. Second, I will utilize engineered DUBs to probe the role of aberrant ubiquitination in distinct subsets of LQT1 trafficking-deficient mutations. Through taking advantage of linkage-specific DUBs and quantitative mass spectrometric analyses, I will probe distinct ubiquitin profiles (i.e. ubiquitin chain types, modified lysine residues) in the trafficking and stability of these mutant channels. In all, this focused, yet innovative proposal will not only contribute significantly to my development as a scientist, but also provide a generalizable toolset to probe the underlying cell biology of ion channels in the heart and illuminate new patient-based therapeutic strategies in inherited arrhythmic disorders. !